Researchers commonly need to extract proteins for a wide range of downstream assays. Total protein extraction is not a complex or challenging technique. But for some special purposes, a researcher may need to obtain proteins from only certain parts of whole cells; such as nuclear proteins. Use of nuclear extracts may be essential for some gene regulation studies. Extracting different fractions from whole cell lysates may be quite challenging. However, there are some nicely designed kits for these purposes. We have used Pierce’s NE-PER nuclear and cytoplasmic extraction kit. This kit is designed for extracting 50 cell pellet fractions. It includes cytoplasmic extraction reagent I (CER I), cytoplasmic extraction reagent II (CER II) and nuclear extraction reagent (NER). The kit should be stored at 4ºC.
The NE-PER Reagents first lyse the cell membrane while maintaining the nuclear compartment thus, recovering the nucleus intact. And then you extract nuclear proteins separately. Nuclear extracts and cytoplasmic protein fractions can be used directly in a variety of downstream applications such as Western blotting, reporter gene assays, enzyme activity assays, and gel shift assays. The kit is convenient for extractions from tissue.
The kit does not support using a protease inhibitor. However, I would recommend using it. You should add the protease inhibitor to CER I and NER from concentrated stocks. To begin with, you may use different volumes of packed cell volumes; you should adjust the substrate volumes carefully. The manual provides a table for this adjustment.
First you obtain the cell pellet, all centrifugation steps must be at 4ºC. After discarding the supernatant carefully, add CER I to the cell pellet. Then vortex the tube vigorously for 15 seconds in order to fully resuspend the cell pellet. Incubate the tube on ice for 10 minutes. The next step is adding ice cold CER II to the mix. Again, vortex the tube for 5 seconds and incubate on ice for one minute. After vortexing for another 5 seconds, centrifuge the tubes for 5 minutes at full speed in a microcentrifuge. Then immediately transfer the supernatant to a clean, pre-chilled tube. This supernatant contains cytoplasmic extract; the pellet contains intact nucleus. Resuspend the pellet in ice-cold NER, then vortex vigorously and put on ice. Continue vortexing for 15 seconds every 10 minutes for a total of 40 minutes, then centrifuge. This time, the supernatant contains nuclear proteins; transfer the supe to a clean tube. As expected, you should store all extracts at -80ºC.
Cytoplasmic and nuclear extracts can be used for many downstream assays. Since your protein may be in present in low concentrations in the cell, I recommend dialyzing the nuclear extract in order to concentrate the nuclear extract. Especially if you encounter problems in downstream assays, you should remove excess salt by dialyzing. The detergent in the reagents, however, is not dialyzable. These detergents will be primarily in the cytoplasmic fractions.
The procedure is very easy. It is a three-step procedure: Lyse the cell, recover the nucleus intact, extract nuclear proteins. It takes little time, generally less than one and a half hours. We have used this reagent for some cancer cell lines, but we have not yet used it with tissue as starting material. We have performed Western blotting with the extracted proteins and did not encounter any problems regarding protein quality in these experiments. In my opinion, the NE-PER Nuclear and Cytoplasmic Extraction Reagents are valuable for obtaining both cytoplasmic and nuclear proteins from the same set of transfected cells. The yield is compatible for many downstream assays.